The Genetic Incorporation of a Distance Probe into Proteins in Escherichia coli

摘要

The unnatural amino acid p-nitrophenylalanine (pNO_2-Phe) is a useful biochemical probe of protein structure and activity.When introduced into proteins either chemically or by in vitro biosynthesis,the pNO_2-Phe group can quench the intrinsic fluorescence of nearby tryptophan (Trp) residues.The ability to genetically encode this amino acid in vivo would greatly facilitate the generation of pNO_2-Phe mutants for both biochemical and cellular studies of protein structure and function.Previously,it was shown that unnatural amino acids can be site-specifically introduced into proteins in bacteria and yeast in response to unique triplet (nonsense) and quadruplet (frameshift) codons.Here we use this methodology to genetically encode pNO_2-Phe in bacteria with high fidelity and efficiency.In addition,we illustrate the use of the pNO_2-Phe/Trp pair as a distance probe in a coiled-coil protein.